Cellulose degradation and utilization

In examining the prospects for a simple process for the degradation and
utilization of cellulose, we chose to look at the thermophilic anaerobic
bacterium, Clostridium thermocellum. This organism grows well at temperatures of
60 C and above. Most important, it has been observed that this organism has the
unique capability to accumulate sugars while degrading and growing on cellulose
(10). This phenomenon is illustrated in Figure 2. In this example, medium
containing 11 g per litre of solka-floc was inoculated with C. thermocellum.
During the course of the fermentation, there is a rapid accumulation of reducing
sugars approximately in parallel with growth and cellulose (expressed as CMCase)
production From the degradation of approximately 10 g/l of cellulose over the
course of 60 hours, there is an accumulation of almost 7 g/l of reducing sugars.
Thus, one achieves approximately a 65 per cent yield of reducing sugars from the
cellulose that is hydrolyzed

When the sugar products are examined by means of high pressure liquid
chromatography (HPLC) (Figure 3), the predominant products are glucose and
cellobiose. When C. thermocellum is grown on natural cellulosic materials
containing hem-cellulose, there is also an accumulation of xylose and possibly
xylobiose. This observation is particularly interesting, because C. thermocellum
will not use the pentoses for growth.

Figure. 3. Accumulation of Glucose and Cellobiose
during In Vivo Saccharification of Cellulose by Clostridium thermocellum

We examined both cellulase and xylanase activity in cell-free broths from C.
thermocellum grown on a variety of substrates, as shown in Table 2. When C.
thermocellum was grown on these substrates, the ratio of xylanase to CMC's
activity was consistently one. These results suggest that cellulose activity and
xylanase activity are caused by the same enzymes. This observation has been made
in other organisms (11), and the dual activity of the cellulase enzymes would
account for the simultaneous accumulation of pentoses and hexoses.

TABLE 2. Production of Xylanase and Cellulose Activity by C. thermocellum
Grown on Selected Carbon Sources

Xylanase*

mg/ml/hr

CMCase*

mg/ml/hr

Xylanase

CMCase

Substrate

Sorka-floc

7.0

6.75

1.04

Corn stover

1.95

1.9

1.03

Avicel

3.0

3.2

0.94

Cotton

4.8

5.0

0.96

* The variation in the values among the substrates reflects the time when the
fermentations were ended, as well as C. thermocellum's ability to grow and
produce enzymes on the particular substrate.

In an attempt to improve and optimize the process, we examined the influence
of pH control during the course of the fermentation. In the previous
experiments, pH was set initially at 6.8 and then left to fall during the course
of the fermentation. Results shown in Table 3 illustrate the difference in
performance for C.thermocellum with controlled pH (6.8) and uncontrolled pH
fermentations on cellulose. As seen by the results in this table, with pH
control there is greater degradation of cellulose, increased cell mass
formation, increased synthesis of fermentation products, e.g., ethanol and
acetic acid, but markedly less accumulation of reducing sugar. These results
suggest that the marked sugar accumulation during growth on cellulose results at
least in part from a restriction in growth, probably by the decreased pH, in the
presence of excess cellulase capacity. Hence, cellulose is hydrolyzed at a rate
faster than it can be utilized.

TABLE 3. Comparison of Results from pH-Controlled and Non-pH-Controlled
Fermentations of C thermocellum Grown on Solka-Floc

Non-pH -controlled

pH-controlled

Initial

Final

Initial

Final

pH

6.8

5.7

6.8

6.8

Cellulose, g/l

10.1

2 4

10.0

1.8

Dry cell weight, g/l

-

0.5

-

0.8

Reducing sugar, g/l

0.3

6.0

0.1

2.4

Ethanol, g/l

-

0.4

-

1.3

Acetic
acid, g/l

-

1.0

-

2.7

The approach illustrated by the above results offers the opportunity for in
vivo saccharification of ligno-cellulosic materials to accumulate sugars, as
well as the possibility for a direct fermentation for product formation from
cellulose. For example, C. thermocellum will produce ethanol, acetic acid, and
lactic acid directly from cellulose. Product accumulation is shown in Figure 4,
which presents results from C. thermocellum grown on cellulose added
intermittently during the course of fermentation. This allowed an increased
amount of cellulose to be acided to the broth. A total of 20 9 of cellulose were
added, and this resulted in the production of 8.5 g/l of reducing sugars and 4
g/l each of ethanol and acetic acid.

The above example illustrates the direct conversion of cellulose to ethanol.
Through genetic manipulation, it is possible to minimize the amount of acetic
acid, which is usually produced in molar ratio of 1:1 with ethanol, so that the
ethanol-acetic acid ratio becomes 8:1 (12). This is an example of what one can
do with high technology to manipulate the ceil in order to generate a cell line
with some desired properties, in this case, over-production of ethanol directly
from cellulose. One of the bottlenecks in this fermentation is the sensitivity
of C. thermocellum to ethanol and aceitic acid. A eel) line that is tolerant to
ethanol concentrations of approximately 5V per cent has been developed, and thus
we have again modified this cell line to achieve a desired final objective
(12).